The goal of the proposed research is the development of reliable and widely applicable methods for the multiplexed in vitro detection of protein biomarkers at extremely low (femtomolar) concentrations in microliter sample volumes. These novel ultrasensitive microarray biosensing measurements will be achieved through the combined use of surface enzyme chemistries, biofunctionalized nanoparticles, and unique surface plasmon-based bioaffinity adsorption detection techniques. Key components in these new methodologies are (i) the use of enzymatic reactions for the attachment, modification and amplification of biomolecules on both nanoparticle surfaces and microarray elements to increase the selectivity and sensitivity of the bioaffinity sensing process, (ii) the incorporation of chemically and enzymatically biofunctionalized nanoparticles into the microarray sensing measurements in order to enhance the capture of and signal generated by target biomolecules, and (iii) the implementation of novel optical detection methods such as surface plasmon resonance phase imaging and nanoparticle-enhanced optical diffraction that provide additional biosensing sensitivity in a multiplexed format. A combination of aptamers, antibodies and enzymatic attachment reactions will be used to capture and detect protein (e.g., VEGF, PSA, troponin) and microRNA biomarker targets that exist in biofluid samples at zeptomole levels. These new detection methodologies that can identify and quantitate multiple proteins at femtomolar concentrations and below are essential technologies for the discovery of new sets of protein biomarkers, and the ability to track these new biomarkers (e.g., cardiovascular and cancer biomarkers) at such low levels will facilitate the creation of better methods for early disease detection and strategies for post-treatment patient monitoring. PUBLIC HEALTH RELEVANCE: Novel methodologies will be created and demonstrated for the simultaneous, multiplexed detection and identification of extremely small amounts (down to thousands of molecules) of biologically relevant proteins and nucleic acids, also known as "biomarkers". These ultrasensitive biosensing technologies are essential for the discovery of new sets of protein biomarkers that are only present at minute concentration levels in biological fluid samples, and will facilitate the creation of new methods for earlier and more precise disease detection (e.g., cancers, heart disease), as well as the creation of better post-treatment patient monitoring strategies.